1. Field of the Invention
This invention relates to a method of clinical diagnosis, more particularly, to a method for quantitatively determining the enzymatic activity of glucoamylase present in urine or body fluids using maltotriitol, phenyl-.alpha.-glucoside or 2,4-dinitrophenyl-.alpha.-glucoside, based on the substrate specificity of glucoamylase without being influenced by the presence of other amylases.
2. Description of the Prior Art
It was found by one of the present inventors that glucoamylase is usually present in urine (Minamiura, N. et al., J. Biochem. 72, 1295 (1972)).
It is also known that glucoamylase originates in the liver, and a relationship between glucoamylase and disorders of the liver and the other organs has been noted.
Therefore, it has been desired to develop a method for quantitatively determining the enzymatic activity of traces of glucoamylase contained in human urine or body fluids.
Generally, in determining amylase activity a buffer solution is added to an enzyme solution to maintain optimum pH, and a substrate is added thereto to allow the mixture to react at an appropriate temperature. After a prescribed period of time, the amount of reduced sugar degradation product is chemically or enzymatically determined to calculate the enzymatic activity.
It is rare that only one type of amylase is produced in an organism. The amylase always exist as a mixture of several kinds. Further, any of the substrates for which each of the amylases is specific is degraded by the amylases to glucose or polymers or oligomers of glucose, e.g., maltose, isomaltose, cyclic dextrin or the like, and, therefore, it is impossible to correlate the degradation products with one kind of amylase, thereby making it difficult to determine the amount of a specific amylase. In practice, quantitative analysis has been carried out by taking into consideration other properties of the amylases.
Several determination methods are conventionally employed which can be selected depending upon the enzyme source and the enzyme whose determination is desired. These can be classified as follows:
1. Thermally resistant amylases and acid resistant amylases are separated. PA1 2. Saccharogenic amylases and liquefying amylases are separated. PA1 3. The degree of polymerization of the degradation product is determined by an iodine method. PA1 4. A specific dextrin is used as a substrate.
With any of these conventional methods, it is difficult to obtain an exact fractional determination.
Glucoamylase is classified as a saccharogenic amylase which produces only glucose from starch glycogen; a fractional determination method therefor distinguishing it from other amylases present which are also capable of producing glucose has not yet been reported.